Human coagulation factor IXa (fIXa) plays a key role in maintaining internal homeostasis in the intrinsic pathway of the clotting cascade. The importance of fIXa in homeostasis is indicated by the frequency of hemophilia, which afflicts 1 in 30,000 males.
Human coagulation factor IX (fIX) is a 415 residue single chain molecule circulating in the plasma. Factor IX is converted to activated factor IX (fIXa) through the cleavage of two bonds by either factor VIIa or factor XIa, releasing a 35 residue activation peptide. After release of the peptide, factor IXa consists of an N-terminal light chain and a C-terminal heavy chain held together by a disulfide bond. fIXa assembles with factor VIIIa on the surface of endothelial cells or activated platelets forming the intrinsic Xase, a potent activator of factor X.
FIX is a multi-domain protein consisting of an N-terminal γ-carboxyl glutamic acid domain followed by two epidermal growth factor-like repeats, an activation peptide and a C-terminal protease domain with a trypsin-like active site. This multi-domain structure defines the fIX gene family of clotting factors, which also includes fVII, fX and protein C. Within this family, fIXa has unique proteolytic properties. Complex formation of fIXa with fVIIIa on a phospholipid surface increases reactivity against the natural substrate IX 106-fold while virtually no cleavage of peptides with the corresponding fX sequences was observed.
Inhibition of fIXa presents an alternative and viable way of treating thrombosis arising from both venous as well as arterial vascular injuries. It is believed that inhibiting fIXa selectively limits thrombosis at sites with low tissue factor presence, but does not inhibit clotting in high tissue factor environments such as vascular injuries of surgical wounds (McKean, M. L. & Adelman, S. J., Expert Opin Investig Drugs (1998) 7:687; Weitz J. I. & Bates S. M. J. Thromb. Haemost. (2005) 3:1843). Factor IXa-specific inhibitors could provide a choice of anti-coagulants with improved therapeutic index compared to existing therapies which target thrombin.
The provision of factor IXa crystals bound to inhibitor compounds provides, e.g., valuable insight into the requirements for compounds that effectively bind to and inhibit factor IXa, and, thus, factor IXa-dependent clotting events. Such crystals also provide useful tools of the generation of novel factor IXa complexes.